Carbohydrates comprise one of the largest and most diverse collections of biologically-active molecules. However, relative to other biomolecules such as nucleic acids and proteins, carbohydrates remain relatively unexplored, and their structure-function relationships are still poorly understood. The broad objective of this program is to develop chemical approaches to advance a fundamental understanding of the roles of carbohydrates in biology and disease. In the last granting period, we developed chemical methods for the rapid, sensitive detection and study of O-linked -D-N-acetylglucosamine (O-GlcNAc) glycosylation. O-GlcNAc is an abundant, essential post-translational modification that is emerging as a key regulator of many physiological functions, ranging from epigenetic and transcriptional gene regulation to insulin signaling, cancer cell metabolism, and neurodegeneration. Our chemical methods enabled the first proteome-wide analyses of the modification and investigations into the dynamics, stoichiometry, and site-specific functions of O-GlcNAc. In the coming granting period, our goal is to tackle the next set of critical barriers in the field. In Aim 1, we will develop new methods to understand the complex regulation of the O-GlcNAc transferase (OGT) enzyme. Our studies will determine the protein interaction and substrate networks of OGT, how specific structural domains within OGT contribute to those networks, and how cellular stimuli dynamically alter the networks. These studies will address the central question of how a single enzyme regulates so many diverse biological processes. In Aim 2, we will address the critical need for detailed structural and biochemical studies of O-GlcNAc-modified proteins by developing a general chemical method for the semi-synthesis of homogeneously O-GlcNAcylated proteins. In Aim 3, we will follow up on our exciting discovery that glycosylated phosphofructokinase 1 (PFK1) may represent a novel anti-cancer target by developing compounds that modulate PFK1 activity and testing their effects on cancer metabolism and growth. In Aim 4, we will investigate the broader roles of O-GlcNAc in regulating cellular metabolism by studying its role in the PI3K-Akt-mTOR pathway, a pathway frequently deregulated in many cancers. Understanding how O-GlcNAcylation regulates mTOR signaling may provide a novel approach to modulate this critical pathway and lead to the discovery of new strategies for therapeutic intervention. Overall, this project will provide essential new insights into the functions of O-GlcNAc and lead to new methods, novel hypotheses, and biological discoveries that will drive the field forward. In addition, the work is expected to reveal new potential therapeutic targets and/or approaches. Finally, a distinctive aspect of the proposed experiments is the seamless integration of chemistry and biology, which we believe is a powerful combination for obtaining fundamental insights into the structure-function relationships of carbohydrates and for advancing the frontiers of chemical biology, glycobiology, and cancer biology.